In this application it is proposed to target mutations (deletions and point mutations) into the envelope glycoprotein (env) gene of Rous sarcoma virus. Specifically the effect of alterations in the signal sequence, and stop-translocation sequence of this gene will be studied. In addition, attempts will be made to localize sequences important in intracellular translocation by isolating mutants defective in different stages of intracellular movement. For these studies the complete, infectious (by DNA transfection) DNA genome of RSV-SR-A cloned into the Sal I site of the bacterial plasmid pBR-322 will be used as a target for mutagenesis. Targetted deletions will be created by D-loop formation and S1 nuclease digestion; point mutations by gapping and single strand mutagenesis with bisulphite. The effect of mutations on the synthesis of env gene products will be assessed after transfection and virus rescue in susceptible cells. The exact location and nature of mutations will be determined by restriction endonuclease analysis and DNA sequencing. These studies will provide important new information on the primary amino acid sequences that are important in the translocation of a transmembrane protein across cellular membranes: processes that are critical to the normal processing and secretion of an enormous number of cellular products. In addition, the experiments described will enhance our understanding of the synthesis of a structural protein from a group of naturally oncogenic animal viruses that is critical for their infectivity and which may play a role in their oncogenicity.